GoldenMutagenesis

GoldenMutagenesis enables targeted multi-site mutagenesis using Golden Gate cloning to introduce simultaneous alterations at one to five amino acid positions within coding sequences for directed enzyme evolution.

Key Features:

• Multi-site mutagenesis: Enables simultaneous alterations at one to five amino acid positions within a coding sequence.
• Golden Gate cloning: Uses the Golden Gate cloning technique with restriction enzymes that cut outside their recognition domains to assemble PCR-amplified gene fragments seamlessly without altering the open reading frame.
• Modular cloning approach: Assembles multiple gene fragments obtained via PCR amplification into reconstituted genes while preserving reading frames for complex cloning and mutagenesis setups.
• Automated primer design: Provides an R-library to automate primer design for introducing specified mutations.
• Graphical evaluation of sequencing results: Includes computational graphical evaluation of sequencing results to assess the quality and success of randomization and mutagenesis outcomes.

Scientific Applications:

• Directed enzyme evolution: Generation of genetic diversity and targeted exploration of protein function and stability via multi-site mutagenesis.
• Enzyme engineering: Development of enzymes with enhanced or novel properties through simultaneous alterations at multiple residue positions.
• Mutagenesis library generation and screening: Creation of mutagenesis libraries compatible with high-throughput screening workflows to identify improved variants.

Methodology:

An R-library automates primer design and computational graphical evaluation of sequencing results is provided to assess randomization and mutagenesis outcomes.